The cell surface receptor, low-density lipoprotein receptor-related protein 5 (LRP5), is emerging as a key regulator of bone mass and strength. Loss-of-function mutations in LRP5 cause the human disease osteoporosis-pseudoglioma syndrome (OPPG), characterized by severely reduced bone mass and strength. Other mutations in LRP5 have been associated with high bone mass (HBM) disorders, characterized by increased bone mass and strength. Mice engineered with loss-of-function mutations in Lrp5, which model the OPPG condition, have dramatically reduced skeletal responsiveness to mechanical loading both in vivo and in vitro, suggesting that Lrp5 plays a key role in bone cell's ability to respond to loading (e.g., exercise). The goal of the present application is to understand precisely how LRP5 participates in the skeleton's response to mechanical loading, which ultimately could suggest new approaches for preventing or treating common diseases of bone-a primary mission of NIAMS (NIH). Among the questions addressed are 1) What steps in the process of mechanoresponsiveness are affected by loss of LRP5 function and by missense mutations that cause HBM phenotypes? 2) Is mechano-responsiveness mediated by canonical Wnt signaling? 3) What ligands are involved in transmitting the mechanical messages through Lrp5? 4) Which, if any, inhibitory proteins participate in this process? 5) Does Wnt/Lrp5 signaling in osteoblast-lineage cells modulate resorption signaling during disuse or overuse? The proposed project is a collaboration between two skeletal biology labs (Indiana Univ. and Case Western Reserve Univ.), which contribute complementary expertise that will facilitate the elucidation of Lrp5's role in bone mechano-responsiveness at multiple levels. In vitro mechanical loading and unloading studies will be conducted using several mouse models, including OPPG (Lrp5-/-) mice crossed with several reporter strains, and 2 HBM mutant strains, in order to determine the mechanisms of Lrp5's effect on mechano-responsiveness. To more fully dissect the role of Lrp5 in mechanical signal transduction, primary osteoblasts will be harvested from these mice and mechanically stimulated in vitro. Specifically, we will investigate (Aim 1) the role of Lrp5 in load-induced osteoblast lifestages (origin, recruitment, differentiation, and fate) and in load-induced canonical Wnt signaling; (Aim 2) where Lrp5 activity occurs in the mechanotransduction signaling cascade, including identification of upstream modulators and downstream signal transduction target pathways; (Aim 3) the role of Lrp5 in regulating mechanically-induced expression of pro-resorption markers, including OPG and RANKL; and (Aim 4) the mechanism of action by which two HBM mutations modulate mechanotransduction. Insights into the mechanisms of Lrp5 activity in mechano-responsiveness hold great potential in the public health arena for understanding the bone-building effects of loading on bone mass, fragility, and fracture risk.